The present invention is generally in the field of xenotransplantation, and genetic modification of animals to produce tissue, cells or organs less likely to induce rejection following transplantation.
Shortage of Organs for Transplantation
As reviewed by Dorling, A., et al., Clinical xenotransplantation of solid organs. Lancet 349:867–871, 1997, clinical transplantation has evolved over the last forty years to the point that organ allografts (i.e., transplants from one animal into another animal of the same species, such as human to human) are a routine treatment option for end-stage kidney, heart, lung, liver and other organ disease. However, there are not enough cadaveric organs to meet the clinical demand. Xenografts (i.e., transplants from one animal into another animal of a different species, such as from a pig into a human) provide a means for keeping end-stage patients alive, either permanently or temporarily until a suitable allograft can be obtained. Ideally, xenografts will be developed using genetic engineering of non-primate species that are suitable for long-term replacement of damaged or diseased organs and subject only to minimal rejection. However, the organs are still useful even if subject to some form of rejection by the new host. Even in the case of allografts, rejection frequently develops and so patients are immunosuppressed using drugs such as cyclosporine and other types of immunosuppressants to prevent rejection of the allograft.
One solution to the problem of organ supply would be the use of organs taken from a suitable animal donor. Although the higher nonhuman primates (apes and Old World monkeys) would provide the closest immunological match for humans, there are several factors that make the routine use of these species as organ donors unlikely. These include (i) inadequate numbers, (ii) difficulty and expense of breeding in large numbers, (iii) inadequate size of some organs (e.g., heart) for adult humans, (iv) probability of public concern regarding the use of such species for this purpose, and (v) risk of transfer of serious viral disease.
Attention is, therefore, being directed towards more commonly available mammals that are lower on the phylogenetic scale, in particular, the pig, which has many advantages in this respect, as reported by Kirkman, R. L. In Xenograft 25, Amsterdam, Elsevier, 1989. pp. 125–132; and by Cooper, D. K. C., et al. In Xenotransplantation. Heidelberg, Springer, 1991. pp. 481–500. These include (i) availability in large numbers, (ii) inexpensive to breed and maintain, (iii) suitable size for the smallest or largest of humans, (iv) availability of pathogen-free (gnotobiotic) animals, (v) considerable similarities of anatomy and physiology with humans, and (vi) ability to genetically engineer.
Shortage of Safe Blood for Transfusion
Eleven million blood transfusions utilizing packed human red blood cells (RBCs) are administered in the US each year (National Blood Data Source, 1998). The U.S. blood supply is chronically inadequate. In 2001, it is anticipated that U.S. Blood Banks will obtain about 250,000 units less than optimally required. Officials are forecasting a critical national shortage during the summer months, when regular blood donors go on vacation and college students also leave the major urban centers. Because the nation has a robust and competitive blood collection and distribution system, periodic shortages of blood do not usually result in deaths, but elective surgeries may need to be postponed and other noncritical needs are not met. As donated blood can be stored under normal conditions for only approximately 42 days, and as less than 5% of eligible donors give blood, severe weather conditions, e.g. snowstorms or hurricanes, by reducing access of potential donors to the Blood Center, often lead to the cancellation of elective surgeries.
Not only is human blood a scarce resource, it also comes with a potential risk to the recipient. Despite new viral screening processes, donated human blood is not considered to be 100% safe. It is estimated that the hepatitis C virus is transmitted once in every 100,000 transfusions, and HIV (the AIDS virus) once in every 676,000. This significant incidence of HIV, hepatitis and other viral agents, particularly in some populations, makes it costly and difficult to provide sufficient safe human blood for purposes of transfusion. More recently, because of concerns over the increasing incidence of new variant Creutzfeldt-Jacob Disease in Europe, eligibility for blood donation has been made more restrictive by the FDA. This may further impact the availability of blood in the USA.
Because of the difficulty and expense of ensuring that human blood is free of any infectious microorganisms, it would be highly desirable to develop a source of RBCs that would be both unlimited in quantity and free of all infectious agents. Pig red blood cells (pRBCs) could fulfill this role.
Although there has been tremendous interest in developing blood substitutes such as perflurochemicals and hemoglobin derivatives, formidable hurdles have been encountered in clinical trials. As reported, an unexpectedly high number of deaths among patients with trauma led to the termination of clinical trials and the withdrawal of two hemoglobin-based formulations from further development (Sloan, E. P., et al., JAMA. 1999: 282: 1857–64). The current absence of a suitable alternative to human RBCs increases the potential importance of pRBCs.
Rejection of Xenografts
Survival of pig-to-human (or other primate) organ or cell transplants is currently limited, however, initially by a severe humoral immune response (hyperacute rejection) that leads to destruction of the graft within minutes or hours, as reviewed by Taniguchi, S. & Cooper, D. K. C. Ann. R. Coll. Surg. Engl. 79, 13–19, 1997; and Cooper, D. K. C., et al. J. Heart Transplant 7:238–246, 1988, and subsequently by a delayed humoral response (acute humoral xenograft rejection) that is believed also to be mediated largely by the effect of anti-pig antibodies.
Xenotransplants between closely-related species (e.g., chimpanzee-to-human) can usually survive the initial period of blood perfusion without damage, as do allotransplants. Subsequently, the foreign antigens of the transplanted organ trigger the recipient's immune response and the rejection process begins. These xenografts, which are rejected clinically rather like allografts, but in an accelerated manner, are termed concordant xenotransplants.) Xenografts between phylogenetically more distant species (e.g., pig-to-human) follow a clinical course quite different from allotransplants and are termed discordant xenotransplants. In discordant xenografted organs, antibody-mediated (vascular) rejection generally occurs within a few minutes or hours of recirculation, with a typical histopathological pattern of endothelial lesions with severe interstitial hemorrhage and edema. This hyperacute rejection is usually irreversible, but can be delayed by removal of the recipient's natural antibodies against the donor tissue. There is now considerable evidence to suggest that this hyperacute rejection is entirely or largely a result of antibody-mediated complement activation through the classical pathway, as reported by Paul, L. C. in Xenotransplantation Heidelberg, Springer, 1991. pp. 47–67; and Platt, J. L., Bach, F. H. In Xenotransplantation, Heidelberg, Springer, 1991. pp. 69–79. Much attention has been directed towards inhibiting this humoral response, as described by Cooper, D. K. C., et al. Immunol. Rev. 141, 31–58, 1994; by Cooper, D. K. C., et al. Xenotransplantation 3, 102–111, 1996; and by Alwayn, I. P. J., et al. Xenotransplantation. 6, 157–168, 1999.
The Gal Antigen-Anti-Gal Antibody Interaction
Studies have shown that there are certain carbohydrate structures present on the surface of mammalian cells, with the exception of Old World monkeys and apes, that elicit an antibody-mediated rejection immediately following implantation of the cells into humans. The antibodies are pre-existing—that is, they are present in the patient's blood prior to implantation of the xenograft—which is why the humoral, or antibody-mediated, response is so intense and immediate. One carbohydrate structure present in pig but not human that elicits an immune response against the pig tissues when transplanted into humans has previously been identified. This is the Gal epitope. Significant levels of IgG, IgM and IgA anti-Gal antibodies are detected in humans. It is known that the lack of Gal epitopes in humans, apes and Old World monkeys is the result of a mutation in the gene for the enzyme, α1,3galactosyltransferase (α1,3 GT) (Larsen, R. D., et al, J. Biol. Chem. 265, 7055–7061, 1990). Several approaches have been suggested to prevent the hyperacute rejection resulting from binding of human anti-Gal antibodies to pig Gal antigens:
(1) “Knock out” of the gene encoding the enzyme, α1,3GT, required for the production of Gal (Thall, A. D., et al, J. Biol. Chem. 270; 21437–40, 1995). To prevent expression of α1,3GT, the gene could be deleted, interrupted, or replaced, either within the coding region or within the regulatory sequences, so that the enzyme is not produced. This is generally accomplished by manipulation of animal embryos followed by implantation of the embryos in a surrogate mother. The embryos can be manipulated directly by injection of genetic material into the embryo by microinjection or by vectors such as retroviral vectors, or indirectly, by manipulation of embryonic stem cells. The latter methodology is particularly useful when the desired end result is to completely prevent expression of a gene for an active enzyme. This approach is currently not possible with regard to the pig as porcine embryonic stem cells have not been isolated, though it is likely to become possible using nuclear transfer technology. The animals would be genetically engineered so that they do not make the Gal epitopes on the surfaces of their cells.
(2) Reduction or suppression of α1,3GT gene expression. In some cases, it may simply be that one wants to decrease expression of Gal. Where, for example, there is a role for Gal that is essential to viability or health of the animal, the optimum results may be achieved by reduction or suppression, rather than by elimination, of gene expression. In these cases, one may want to introduce a gene for an enzyme that can compete for substrate with the α1,3GT and thus reduce the number of Gal epitopes (Cooper, D. K. C., et al. Lancet 342, 682–683, 1993). It is possible to reduce the expression of the Gal epitopes on the animal tissues by inserting a gene for an enzyme that competes with α1,3GT for the common substrate, N-acetyllactosamine, thus reducing the immune response following transplantation. The DNA encoding another enzyme for modification of the sugar structures, such as a sialyltransferase or a fucosyltransferase, can be inserted into the embryo where it is incorporated into the animal's chromosomes and expressed to modify or reduce the immunoreactivity of the Gal structures on the cell surfaces. This has been achieved in mice (Osman, N., et al, Proc. Natl. Acad. Sci. USA, 94, 14677–14682, 1997; Shinkel, T. A., et al. Transplantation 64, 197–, 1997; Tanemura, M., et al. Transplant. Proc. 29, 895, 1997) but to date has been only partially successful in pigs (Koike, C., et al, Xenotransplantation 3, 81–86, 1996; Sharma, A., et al. Proc. Natl. Acad. Sci. USA 93, 7190–7195, 1996), and has been reviewed by Cooper, D. K. C. Xenotransplantation 5, 6–17, 1998.
It is preferable to modify the epitope to a carbohydrate that is present in the human subject so that antibodies against this carbohydrate are not present in the human recipient of the animal organ. If it is modified to any other carbohydrate, then antibodies to this carbohydrate might develop if the carbohydrate is not naturally occurring in the human subject. This may be achieved by genetically engineering the animals which serve as the source of the xenografts to express either a sialyltransferase or fucosyltransferase so that nonGal carbohydrate structures (that are also present in humans) are attached to the substrate (which is usually used for the formation of Gal epitopes) to prevent recognition and binding by the naturally occurring anti-Gal antibodies (Osman, N., et al, J. Biol. Chem. 271, 33105–33109, 1996; Osman, N., et al. Proc. Natl. Acad. Sci. USA, 94, 14677–14682, 1997; Sandrin, M. S., et al. Xenotransplantation 3; 134–140, 1996; Sandrin, M. S., et al. Nature Med. 1, 1261–1267, 1995). A human α-1,3 fucosyltransferase has been cloned by Koszdin & Bowen, Biochem. Biophys. Res. Comm. 187, 152–157, 1992; and by Lowe, J. B., et al., J. Biol. Chem. 266, 17467–17477, 1991.
(3) Insertion of the gene for α-galactosidase that deletes terminal Gal residues, thus reducing Gal expression (Cooper, D. K. C., et al, Xenotransplantation 3, 102–111, 1996; Osman, N., et al, Proc. Natl. Acad. Sci. USA 94, 14677–14682, 1997);
(4) Immunoadsorption of anti-Gal antibodies from the primate recipient. Human serum contains anti-pig antibodies, which include anti-Gal IgG, IgM and IgA (Good, A. H., et al, Transplant. Proc. 24, 559–562, 1992; Cooper, D. K. C., et al, Transpl. Immunol. 1, 198–205, 1993; Kujundzic, M., et al. Xenotransplantation. 1, 58–65, 1994). Anti-Gal antibodies can be removed from human plasma by plasma exchange or adsorbed by passing the plasma through an immunoaffinity column of one or more of the specific Gal structures. The adsorption of such anti-pig antibodies by the specific Gal carbohydrate can prevent the hyperacute rejection that occurs when xenotransplantation is carried out between pig and a nonhuman primate, as reported by Ye, Y., et al. Transplantation. 58, 330–337, 1994; by Cooper, D. K. C., et al. Xeno. 4, 27–29, 1996; and by Xu, Y., et al. Transplantation. 65, 172–179, 1998.
(5) The intravenous administration of one or more Gal carbohydrates (e.g., synthetic Gal oligosaccharides) that would be bound by the endogenous antibodies and thus prevent binding to the xenotransplant (Ye, Y., et al, Transplantation 58, 330–337, 1994; Simon, P., et al. Transplantation 65, 172–179, 1998; Romano E., et al, Xenotransplantation 6, 36–42, 1999).
There is increasing evidence to suggest that the same approaches (1–5, above) will prevent or delay the development of acute humoral xenograft rejection, which is the rejection response that develops if hyperacute rejection has been prevented or avoided (Alwayn, I. P. J., et al. Xenotransplantation. 6, 157–168, 1999; and reviewed in Buhler, L., et al. Frontiers in Bioscience 4, d416–432, 1999. http://www.bioscience.org/1999/v4/d/buhler/fulltext.htm (Pub med identification number 10209058).
Methods to create pigs, as well as other animals, for use as potential organ and tissue donors, have been developed based on this information. Once genetically engineered animals are produced, tissues, including skin, heart, livers, kidneys, lung, pancreas, small bowel, and components thereof are harvested and can be implanted as known by those skilled in the art of transplantation.
However, all of the above approaches, while useful, have yet to prove fully successful or have not completely solved the problems of antibody-mediated xenograft rejection.
It is therefore an object of the present invention to provide a solution to the problem of alleviating immune rejection of xenotransplants, specifically pig into human, where the rejection is initiated by the presence of glycoprotein and/or glycolipid structures on the vascular endothelium of the xenotransplant which are not found in the human.
It is a further object of the present invention to provide genetically engineered cells, tissues and organs that do not express specific sugars (Gal and NeuGc) which may elicit an immune response, including a complement-mediated response, following transplantation of an animal organ, tissues or cells into a human.
It is a still a further object of the present invention to provide a means for providing animal RBCs that can be transfused into humans without adverse reactions.
It is yet another object of the present invention to provide a means for producing therapeutic glycoproteins without specific immunogens (Gal and NeuGc) to prolong the in vivo half-life of these glycoproteins in humans.